EP2089526B1 - Auf die expression und die kontrolle der posttranslationalen modifikationen eines rekombinanten polypeptids gerichteter satz von sequenzen - Google Patents

Auf die expression und die kontrolle der posttranslationalen modifikationen eines rekombinanten polypeptids gerichteter satz von sequenzen Download PDF

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EP2089526B1
EP2089526B1 EP07859279A EP07859279A EP2089526B1 EP 2089526 B1 EP2089526 B1 EP 2089526B1 EP 07859279 A EP07859279 A EP 07859279A EP 07859279 A EP07859279 A EP 07859279A EP 2089526 B1 EP2089526 B1 EP 2089526B1
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protein
gfp
golgi
polypeptide
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EP2089526A2 (de
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Véonique GOMORD
Claude Saint-Jore-Dupas
Aurelia Boulaflous
Marie-Christine Kieffer-Meyer
Loïc FAYE
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Centre National de la Recherche Scientifique CNRS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8221—Transit peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon

Definitions

  • the present invention relates to the field of recombinant proteins production and is more particularly related to methods for producing recombinant polypeptides that are post-translationally modified in the endoplasmic reticulum (ER) and/or the Golgi apparatus (GA).
  • the present invention provides tools useful for controlling the post-translational modifications of recombinant polypeptides and more generally DNA manipulation tools for plant genetic modification.
  • the present invention also provides processes for producing a recombinant polypeptide involving these tools.
  • the tools of the present invention include targeting signals allowing the sorting of recombinant polypeptides during their synthesis in a host cell to specific sub-cellular compartments and allowing also a specific designing of said recombinant polypeptides within said sub-cellular compartments.
  • the present invention allows advantageously, for example, an increase of the yield of production of recombinant polypeptides, a limitation or prevention of immunogenicity of the recombinant polypeptides and obtaining therapeutically active recombinant polypeptides that are the exact copy of their natural counterpart.
  • the present invention relates particularly to the field of reorientation of plants made pharmaceuticals (PMP).
  • the present invention relates also to the field of immune targeting of plant made pharmaceutical using protein interaction with Single Chain variable fragment (ScFv) fused with targeting signals.
  • ScFv Single Chain variable fragment
  • Recombinant DNA technology has enabled the production of heterologous recombinant proteins in host systems.
  • the majority of the early work was directed toward the expression of recombinant therapeutic proteins in prokaryote hosts, mainly in Escherichia coli.
  • the advantages of prokaryotes as a production system are the ease with which they can be manipulated genetically, their rapid growth and high expression level of recombinant proteins and the possibility of a large-scale fermentation.
  • Plant cells and plant suspension-cultured cells can represent a good alternative : advantageous costs, no pahogenic contamination against human. ( Gomord et al. 2004 (ref 23))
  • PTMs Post Transcriptionnal Modifications
  • PTM covers covalent modifications that yield derivatives of individual amino-acid residues for example glycosylation, phosphorylation, methylation, ADP-ribosylation, oxidation and glycation; proteolytic processing by reactions involving the polypeptide backbone; and non-enzymatic modifications, such as deamidation, racemization and spontaneous changes in protein conformation.
  • the secretory pathway is composed of the endoplasmic reticulum (ER), the Golgi apparatus (GA), the tonoplast, the lysosomal compartments, the plasma membrane and the extracellular medium as represented in annexed figure 1 .
  • ER endoplasmic reticulum
  • GA Golgi apparatus
  • tonoplast the endoplasmic reticulum
  • lysosomal compartments the plasma membrane and the extracellular medium as represented in annexed figure 1 .
  • the early compartments ER and GA
  • the proteins are subcompartimented in four domains enzymatically distincts in the plant cells (see figure 1 : domains blue, yellow, green and orange).
  • therapeutic proteins including blood proteins, cytokines, immunoglobulins, structural proteins, (growth) hormones, vaccines, enzymes and lysosomal proteins, are co-translationally inserted in the lumen of the ER, and then transported via the GA to the lysosomal compartment, the extracellular matrix or the blood stream. Most modifications of therapeutic proteins occur in the secretory pathway but in particular in its early compartments (ER and GA, see Gomord and Faye , 2004 (ref 21).
  • coagulation factor IX is a vitamin-K-dependent glycoprotein synthesized as a precursor molecule of 461 amino acids in the ER.
  • this precursor undergoes extensive posttranslational modifications in the ER and in the GA, including the cleavage of a signal peptide and a propeptide, disulfide bridge formation, ⁇ -carboxylation of the first 12 glutamic acid residues, partial ⁇ -hydroxylation of aspartate 64, N-linked glycosylation at Asparagine (Asn) 157 and Asn 167, O-linked glycosylation at Serine (Ser) 63, Ser 61, Threonine (Thr) 159, Thr 169, Thr 172 and Thr 179, sulfation of Tyrosine (Tyr) 155, and phosphorylation of Ser 158.
  • This is one the most complex maturations of a therapeutic protein ever observed.
  • most therapeutic proteins require at least proteolytic cleavage(s) and glycosylation for their bioactivity, pharmacokinetics, stability and solubility.
  • Eukaryotic cells are able to realize most of these modifications. However, these maturations are more generally specific of the host systems. Moreover, post-translational modifications are different from mammalian cell to plant cell. In plants, as in other eukaryotic cells, N-glycosylation starts in the ER, with the cotranslational addition of an oligosaccharide precursor (Glc3Man9GlcNAc2) to specific asparagine residue constitutive of potential N-glycosylation sequences, Asn-X-Ser/Thr.
  • Glc3Man9GlcNAc2 oligosaccharide precursor
  • the oligosaccharide precursor undergoes several maturations involving the removal or addition of sugar residues in the ER and the GA as shown schematically in annexed figure 2 . It is only in the late GA that plant and mammalian N-glycan maturation differs, which results in the absence of alpha-(1,6)- linked fucose, beta (1,4)-linked galactose and sialic acids and the presence of bisecting beta-(1,2)-xylose and core alpha-(1,3)-fucose in the N-glycans of PMPs (see annexed figure 2 ).
  • This strategy consists in the storage of recombinant proteins within the ER, i.e., upstream of Golgi cisternae, where immunogenic glyco-epitopes are added to complex plant N-glycans. It was first shown that the addition of H/KDEL sequences at the C-terminal end of a recombinant soluble protein is sufficient for its retention in the plant ER (Gomord et al., 1997 ( ref . 24), 1999 ( ref . 22), Saint-Jore-Dupas et al., et 2004 ( ref . 57)).
  • the alpha-glucosidase I is the first enzyme involved in the maturation of the N-linked oligosaccharide precursor by removing specifically the distal alpha-(1,2)-linked glucose residue from the oligosaccharide precursor just after its transfer "en block” on the nascent glycoprotein (see Figure 2 ).
  • the function and consequently the location of this type II membrane protein in the ER is essential. Indeed, in mammalian cells, its defect in neonate induce severe generalised hypotonia and dysmorphic features to fatal outcome at age 74 days (De Praeter et al., 2000 ( ref. 14)).
  • proteolytic maturations in the secretory pathway is similar in plant and mammalian cells. These maturations are essential for processing of both endogenous and recombinant proteins, but they also make high-yield production of stable, integral polypeptides a challenging task. These proteolytic maturations also depend on the subcellular compartment where the protein is accumulated (Faye et al., 2005 ( ref . 17.)).
  • pre-proproteins also called “not post-translationally modified” in the following description
  • pre-proproteins including an N-terminal cleavable signal peptide - or pre-region - directing the nascent polypeptide chain to the endoplasmic reticulum, and a regulatory pro(poly)peptide - or proregion - involved in the stabilization, targeting, inhibition and/or folding of the mature protein before its translocation to and processing at the final cellular destination.
  • proteins After removal of their signal peptide by a signal peptidase, proteins are released into the ER lumen to be properly assembled and folded, and then translocated to the GA, and eventually further downstream to the different compartments of the secretory system.
  • proproteins Many plant proteins leave the ER as proproteins, with the proregion being proteolytically cleaved downstream along their route through the secretory pathway. It is the case of many vacuolar proteins bearing a C- or N-terminal cleavable sorting signal, which are removed during or after their transport to the vacuole by specific proteases.
  • seed storage proteins such as 2S albumins and 11S globulins
  • proteins with antimicrobial or antifeedant/antidigestive activity such as pathogenesis-related proteins, chitinases, glucanases, lectins and wound-inducible proteinase inhibitors.
  • proteins with antimicrobial or antifeedant/antidigestive activity such as pathogenesis-related proteins, chitinases, glucanases, lectins and wound-inducible proteinase inhibitors.
  • numerous secretory proteins are first synthesized
  • subtilisin-like proprotein convertases a family of enzymes structurally similar to bacterial subtilisins and yeast kexin.
  • these enzymes usually found in the trans-Golgi network convert a variety of protein precursors to mature proteins, thereby directly or indirectly contributing to the fine control of important processes such as zymogen activation, gene expression, cell cycle, programmed cell death, intracellular protein targeting, and endocrine/neural functions.
  • alpha-glucosidase I is primordial in the accumulation of storage proteins, the formation of protein bodies, cell differentiation and cell wall disruptions during Arabidopsis thaliana embryo development. Without alpha-glucosidase I activity Arabidopsis thaliana seed development is blocked at the heart stage (Boisson et al., 2001 ( ref . 6)) and cell wall biosynthesis is strongly affected (Gillmor et al., 2002 ( ref. 19)).
  • the inventors of the present invention have identified two independent types of signals conferring ER residency on Arabidopsis thaliana glucosidase I. Using various deletions or mutants of this glucosidase fused to GFP, they have shown that full length of A. thaliana alpha-glucosidase I (hereafter named GCSI) is strictly accumulated in the ER, and contains Arg-based motifs located in the cytosolic tail sufficient for targeting reporter to the ER. However, these functional Arg-based signals are not required to localize the full-length GCSI in this compartments and a second signal has been identified in the stem of this membrane bound ER enzyme.
  • GCSI A. thaliana alpha-glucosidase I
  • the inventors of the present invention have identified three independent types of signals conferring GA residency on Arabidopsis thaliana glycosyltransferases.
  • the purpose of the present invention is precisely to provide efficient tools responding to this need.
  • the tools of the present invention are in the form of targeting or retention signals and processes involving these targeting signals.
  • the present invention is generally directed to the modification of the post-translational maturation of recombinant polypeptides by different ways using these targeting or retention signals.
  • the inventors Based on their numerous studies on transport and localization of plant or human enzymes, for example of glycosidases and glycosyltransferases, the inventors have identified different peptidic signals specifically involved in the distribution of the membrane proteins between the ER and the GA, in particular in plant cells.
  • a first aspect of the present invention is to provide particular peptidic signals that allow retention of recombinant polypeptides in specific cell sub-compartment, in particular in plant cells.
  • the sequences and structures of these peptidic signals are described below.
  • the peptidic signals of the present invention are selected from the group consisting SEQ IDn°1, SEQ ID n°4 and SEQ ID n° 8.
  • these peptidic signals may be referred to as "retention signal sequence” or “signal sequence” or “targeting signals” or "peptidic signal”.
  • the retention signal sequences of the present invention specifically target a recombinant polypeptide to the ER and/or the GA compartment membranes, in particular of plant cells. These sequences allow retention of the recombinant polypeptides in the ER and/or in different sub-compartments of the GA or under a membrane bound form in the ER and/or different sub-compartments of the GA.
  • target or “targeting” it is meant that a polypeptide fused with a peptidic signal of the present invention will be localized, i.e. confined, in the ER and/or GA because of this peptific signal.
  • Maturation and stability of a recombinant polypeptide depend directly on the compartment where the polypeptide is accumulated. For instance, the present inventors have shown that retention of recombinant polypeptides in the ER increases their stability and prevents their N-glycan maturation. They have also shown that expression of a soluble recombinant polypeptide as a membrane polypeptide also increases its stability. Finally, they show that retention of a recombinant polypeptide in production of said recombinant polypeptide and may prevents immunogenicity due to glycan maturation.
  • Another aspect of the present invention is to provide recombinant polypeptides comprising a peptidic signal according to the present invention and a polypeptide, said peptidic signal being fused to the polypeptide.
  • the fusion of the peptidic signal of the present invention may be at the C-terminal or N-terminal extremity of the polypeptide.
  • said peptidic signal may be linked to the C-terminal or N-terminal end of the polypeptide or protein.
  • the recombinant polypeptide may be any recombinant polypeptide having an interest in pharmaceutical or agri-food industry.
  • the "recombinant polypeptide” may also be named “recombinant protein” or “peptide X” or “target protein” in the present specification.
  • the polypeptide also named herein "target polypeptide” may be all membrane therapeutical polypeptide, all soluble therapeutical polypeptide that may be expressed as a membrane protein or not, all antibodies and fragments thereof.
  • the recombinant polypeptide may be selected from the group comprising an enzyme, an antibody or part thereof, a reporter protein, a nucleotide transporter and a therapeutically active polypeptide.
  • the recombinant polypeptide is a soluble polypeptide or protein.
  • therapeutically active proteins examples include vaccines, allergens, enzymes, blood proteins, hormone, antibodies, antibody-derived fragments.
  • the therapeutically active polypeptide is soluble.
  • “Therapeutical polypeptide” or “therapeutically active polypeptide” have the same meaning in the present description and claims [or the soluble part of a membrane bound proteins].
  • said enzyme when the recombinant polypeptide is an enzyme, said enzyme may be a plant or an animal enzyme.
  • said enzyme may be selected for example from the group comprising glycosidase, glycosyltransferases, protease, kinase, decarboxylase, epimerase, nucleotide-sugar transporter, for example UDP-sugar transporter, GDP-sugar transporter or CMP-Sugar transporter, amidation enzymes and more generally any maturation enzyme present or not in the ER and/or GA of an host cell, for example a plant cell.
  • Glycosidase may for example be those involved in the N- or O-glycosylation.
  • Glycosyltransferases may for example be those involved in the N- or O-glycosylation.
  • Examples of enzymes are cited in the following table Table of enzymes N-glycosylation Human Beta (1,4) galactosylation Yeast Mannosyltransferase OCH1p Human GNT III O-glycosylation N-acetylglucosaminyl transferase galactosyltransferase Proteolytic cleavage Serine proteases Cyteine proteases amidation Oxygenese lyase Phosphorylation phosphorylase Gamma-carboxylation Gamma carboxylase Proteoglycan modif Glycotransferase Glycosidase Sulfation sulfatase hydroxylation hydroxylase acetylation acetylase Cell wall polysaccharides modif.
  • Glycotransferase Glycosidase Sulfation
  • maturation enzyme it is referred to any enzyme that participate to the maturation of a recombinant protein in a host cell.
  • the protein may be fused with a storage protein or proteins stored in the protein bodies, for example a protein fused to the ZERA®.
  • a storage protein or proteins stored in the protein bodies for example a protein fused to the ZERA®.
  • a further aspect of the present invention is to provide the nucleic acid sequences encoding an amino acid sequence selected from the group consisting of SEQ ID N°1, SEQ ID N°4 and SEQ ID N°8.
  • SEQ ID N°1, SEQ ID N°4 and SEQ ID N°8 examples of nucleic acid sequences encoding SEQ ID NOs: 1-31 are given in the annexed listing sequences under references SEQ ID NOs: 102-132. According to the degenerescence of the genetic code, the skill person will easily deduce other suitable nucleic acid sequences that encode also SEQ ID NOs: 1-31.
  • a further aspect of the present invention is to provide the nucleic acid sequences encoding the recombinant polypeptide according to the present invention.
  • a further aspect of the present invention is to provide nucleic acid vectors comprising a nucleic acid sequence according the present invention.
  • the nucleic acid vector of the present invention comprises a nucleic acid sequence coding for a peptidic signal of the present invention, wherein said nucleic acid is introduced in the vector in frame with the nucleic acid sequence coding for a polypeptide or protein to produce a recombinant polypeptide or protein containing the "retention signal sequence" at one (or both) extremity (extremities) of said polypeptide.
  • any known and suitable method may be use to construct these nucleic acids and nucleic acid vectors.
  • the methods disclosed in (Gomord et al., 1997 (ref. 24) and 1998 (ref. 25), Pagny et al, 2000 (ref. 49) and 2003 (ref. 50), Saint-Jore-Dupas et al 2006 (ref. 58)) may advantageously be used.
  • a further aspect of the present invention is to provide a plant cell comprising at least one peptidic signal of the present invention and/or at least one recombinant polypeptide of the present invention (i.e. including a peptidic signal of the present invention) and/or at least one nucleic acid sequences encoding a recombinant polypeptide of the present invention and/or at least one nucleic acid vector of the present invention.
  • said recombinant polypeptide may be a homologous or a heterologous polypeptide.
  • Recombinant polypeptides may be as defined above.
  • any known and suitable method may be use to obtain said plant cells.
  • the methods disclosed in Gomord et al., 1997 ( ref . 24), 1998 ( ref . 25) , Pagny et al, 2000 ( ref . 49) and 2003 ( ref . 50), Saint-Jore-Dupas et al 2006 ( ref . 58), Saint-Jore et al., 2002 ( ref . 56) may advantageously be used.
  • a further aspect of the present invention is to provide a plant comprising at least one peptidic signal of the present invention and/or at least one recombinant protein of the present invention (i.e. including a peptidic signal of the present invention) and/or at least one nucleic acid sequences encoding a recombinant protein of the present invention and/or at least one nucleic acid vector of the present invention.
  • said recombinant protein may be a homologous or a heterologous protein.
  • Recombinant proteins are as above-defined.
  • the plant may be any suitable plant that allows the production of a recombinant protein.
  • the plant may selected from the group comprising Alfalfa, Arabidospsis thaliana, Nicotiana tabacum, Glycine max, Lycopersicon esculentum, Solanum tuberosum, oriza sativa, zea maize, moss (physcomitrella patens), Lemna minor, Algae (ostreococcus tauri, phaelodactylum), chlamydomonas reinhardtii.
  • the plant cell may be issued or derived from any of these plants.
  • the present inventors provide further a first method for producing a post-translationally modified heterologous polypeptide in host cells that have been transformed with a vector design for the targeted expression of said recombinant polypeptide.
  • This first method is represented schematically in annexed figure 3 , line (A).
  • the present invention has also for subject a method for producing a post-translationally modified polypeptide comprising the steps of:
  • the process may further comprise a step of screening the cells before the step of growing the transfected or transformed cells.
  • Any method of screening known by the skilled person may be used. For example, by direct selection with a microscope, by electrophoresis SDS page, by immunodetection, by membrane transfert, etc.
  • An example of a method usfull for carring out the screening is disclosed for example in document Gomord et al. 1997 (Ref 24).
  • This step of screening allows selecting the cells that have been transfected or transformed according to the present invention. This step leads to a better yield relating to the mofified polypeptide.
  • recombinant polypeptide for designating the recombinant polypeptide to be produced, for example for a pharmaceutical use
  • recombinant protein for designating a protein involved (see explanations below in the second method description) in the maturation process, i.e. post-translational modification, of the recombinant polypeptide.
  • the use of the nucleic acid vector encoding the peptidic signal of the present invention fused with the recombinant polypeptide allows the retention of the recombinant polypeptide freely or under a membrane bound form in the ER and/or different sub-compartments of the GA.
  • Part of the heterogeneity observed on recombinant polypeptides with the prior art methods occurs during the transport and maturation through the Golgi.
  • the retention of the recombinant polypeptides in the ER or in the ER-derived protein bodies or in the early Golgi compartments by the use of the peptidic signal of the present invention may reduce this heterogeneity.
  • a major difficulty is to produce a recombinant protein, which is the exact copy of its natural counterpart.
  • the polypeptide post-translational maturations differ not only from an expression system to the other but also from an organ to the other in the same expression system and from one sub-cellular compartment to the other in a cell constitutive of the same organ.
  • the addition of a peptidic signal to target a PMP to a specific compartment according to the first method of the present invention may solve this difficulty but this structural modification of the recombinant protein leads of course to a non-native protein.
  • the peptidic signals of the present invention is a real tool allowing further to control the maturation of the recombinant polypeptide, by engineering or designing the ER and GA medium of the host cells. This control allows producing recombinant polypeptides that are more stable within the host cell, that are less immunogenic and that tend to exact copies of their natural counterparts. This is very important for the production of therapeutically active proteins, in particular by plants, usable in human therapy.
  • the present invention provides several solutions to design the ER and GA environment. These solutions may be used alone or together (combination). Examples of these solutions are schematically represented in annexed figure 3 , lines (B) and (C). In all of these solutions, the host cell is transfected or transformed with:
  • a further aspect of the present invention is to provide a second method for producing a post-translationally modified recombinant polypeptide comprising the steps of:
  • the process may further comprise a step of screening the cells before the step of growing the transfected or transformed cells. Any method of screening known by the skilled person may be used. Screening methods may be those disclosed above. This step of screening allows selecting the cells that have been transfected or transformed according to the present invention. This step leads to a better yield relating to the mofified polypeptide.
  • the recombinant protein translated in the host cells comprises the peptidic signal of the present invention and this recombinant protein is different to polypeptide to be produced.
  • the recombinant protein play a role in the modulation of the post-translational modification of the recombinant polypeptide to be produced, i.e. it is involved in the maturation process, i.e. post-translational modification, of the recombinant polypeptide.
  • this recombinant protein may be, for example, an enzyme and/or an antibody or part thereof.
  • the recombinant protein when the recombinant protein is an antibody or part thereof, it may be such as recognizing and binding specifically the polypeptide to be produced and/or an antibodies or part thereof recognizing and binding specifically with an enzyme involved in the post-translational modification of said polypeptide.
  • the antibody or part thereof fused with the peptide signal of the present invention will be localized in the ER and/or GA of the host cell.
  • the role of the recombinant protein is here to capture the recombinant polypeptide in the RE and/or GA. So the instant invention provides a method for producing a post-translationally modified heterologous polypeptide by expressing antibody or antibody fragment in the host cells that have been transformed with an expression vector comprising nucleic acid sequences encoding :
  • the present invention allows advantageously to retain a native (not modified by a tag addition consisting of the peptidic signal of the present invention) recombinant protein in the ER and/or the GA of host cells via its binding to a specific antibody or antibody fragment retained in one of these compartments by fusing said antibody or fragment thereof with a peptide signal according to the present invention.
  • ER and/or GA retention peptide signals according to the present invention have been fused to antibodies with specific affinity for the recombinant polypeptide with the ultimate goal to control or to modulate the maturation of recombinant protein.
  • an application of the present invention is to specifically use inactivating antibodies to the ER and/or GA to inactivate specific enzymes located in these compartments.
  • ER and/or GA retention peptide signals according to the present invention have been fused to antibodies with specific affinity for the catalytic domain of ER or GA enzymes with the ultimate goal to inactivate endogenous enzyme to control or to modulate the maturation of recombinant protein.
  • the role of the recombinant protein is here to capture an enzyme involved in the maturation of the recombinant polypeptide.
  • the present invention allows therefore to "immuno-modulate" endogeneous enzymes within the host cell.
  • the second method may further be used in order to :
  • the capacity of a heterologous or homologous enzyme to modify the maturation of recombinant protein will depend on its localization in the cell. Then, in order to modify the enzymatic equipment of a host cell, it is preferable to target the appropriate enzyme in the "good" compartment. So the present invention provides a method for producing a post-translationally modified heterologous polypeptide by expressing maturation enzymes in the host cells that have been transformed with (an) expression vector(s) containing nucleic acid sequences encoding:
  • said recombinant protein may be an endogenous or heterologous enzyme involved in the post-translational modification of said polypeptide.
  • the present invention allows modifying the enzymatic equipment of the ER and/or the GA in the host cell, in particular in plant cell, according to two separate but juxtaposable strategies: (a) by reorientation of an endogeneous enzyme to ER and/or GA, and (b) by targeting an heterologous enzyme to ER/GA which can be located in different sub-cellular compartments.
  • This modification of the enzymatic equipment of the ER and/or the GA in the host cell is a tool allowing modifying (i.e. designing) the post-translational maturation of the recombinant polypeptide.
  • this modification may allow to improve the stability and/or to control the immunogenicity of the produced recombinant polypeptide.
  • the role of the recombinant protein is here to capture homologous or heterologous enzymes involved in the maturation of the recombinant polypeptide in the ER and/or GA.
  • the present invention allows therefore advantageously getting a recombinant polypeptide that is a non-immunogenic glycoprotein but also a recombinant polypeptide that is a homogeneous glycoproteins.
  • the polypeptide may be any polypeptide needed to be produced by genetic recombination, in particular in plant cells or whole plants.
  • the polypeptide that may be produced with the present invention are for example those cited above in the disclosure of the recombinant polypeptide of the present invention.
  • the first and second method may be used simultaneously.
  • the vector nucleic acid vector encoding said polypeptide is also a nucleic acid vector according to the present invention, i.e. encoding the polypeptide fused with the peptidic signal of the present invention.
  • one may target the recombinant polypeptide for maturation for localization in the ER and/or GA and, in the same time design the ER and/or GA for the maturation, i.e. post-translational modification of the recombinant polypeptide.
  • the polypeptide may be as defined above. For example, it may be a therapeutically active protein.
  • the post-translational modification of the polypeptide is advantageously carried out in the ER and/or GA compartment membranes.
  • the present inventors are the very first ones to provide such powerful tools for producing designed recombinant polypeptides in a host cell, particularly in a plant cell.
  • the polypeptide may be co-expressed with a storage protein.
  • This storage protein may be for example a protein fused to ZERA® or any other suitable protein.
  • Annexed figure 3 , line (D) represent schematically the targeting of ZERA®-recombinant protein fusion to the Golgi.
  • protein bodies may be initiated from the ER membrane by a ZERA®-recombinant protein expression in the host cells (see annexed figure 5A ).
  • Recombinant glycoproteins accumulated in the ER harbour exclusively high mannose type N-glycan.
  • the present invention provides the peptide signals that allow advantageously combining ER homogeneity and glycan modification, for example for a pharmaceutical glycoprotein, stored in the ER after fusion with a ER retention signal.
  • the cells are preferably plant cells, for example as defined above, e.g. plant cells are cells issued from a plant selected from the group comprising Medicago sativa, Arabidopsis thaliana, Nicotiana tabacum, Glycine max, Lycopersicon esculentum, Solanum tuberosum Oriza sativa, Zea mays, Physcomitrella patens, Lemna minor, Ostreococcus tauri, Phaelodactylum.
  • plant cells are cells issued from a plant selected from the group comprising Medicago sativa, Arabidopsis thaliana, Nicotiana tabacum, Glycine max, Lycopersicon esculentum, Solanum tuberosum Oriza sativa, Zea mays, Physcomitrella patens, Lemna minor, Ostreococcus tauri, Phaelodactylum.
  • Figure 1 is a schematic representation of plant cell secretory pathway. The domains of the ER and Golgi continuum are indicated as follows:
  • Figure 2 shows transfert and processing of N-linked glycans on the protein, in the endoplasmic reticulum and Golgi apparatus of plant and mammalian cells.
  • a precursor oligosaccharide assembled onto a lipid carrier is transferred on specific Asn residues constitutive of the nascent polypeptide.
  • the N-glycan is then trimmed off with removal of glucosyl and most mannosyl residues. Differences in the processing of plant and mammalian complex N-glycans are late Golgi maturation events.
  • the ER and Golgi domains are indicated on main arrow as described in Fig 1
  • Figure 3 shows potential applications of different signals (in dark) in the targeting of recombinant protein (line A), of antibodies or part thereof (ScFv) (line B), of Enzymes (line C) or of Zera®-Fusion (line D).
  • Figure 4 is a schematic representation of analyzed fusion proteins:
  • Figure 5 shows localization into the Golgi and/or the ER of a series of GFP fusions to four different members of the N-glycan processing machinery ( ⁇ -glucosidase I, mannosidase I, N-acetylglucosaminyltransferase I, ⁇ -1,2-xylosyltransferase, figure 4 ) and to the C-terminal HDEL ER retention sequence, after stable expression (3-4 days) in tobacco BY-2 cells, using confocal laser scanning microscopy:
  • Figure 6 shows localization into the Golgi and/or the ER of a series of GFP fusions to truncated members of the N-glycan processing machinery (Man99, Man49, GNT38, XylT35, figure 4 ), after stable expression (3-4 days) in tobacco BY-2 cells or transient expression (5 days) in tobacco leaf epidermal cells by leaf infiltration, using confocal laser scanning microscopy:
  • Figure 7 shows localization into the Golgi stacks of a series of GFP fusions to ManI, Man99, Man49 or GNT38 ( figure 4 ) and the trans Golgi marker ST52-mRFP, after stable co-expression (3-4 days) in tobacco BY-2 cells of one or other of these GFP fusions and ST52-mRFP, using confocal laser scanning microscopy:
  • stacks often appear "tri-coloured” (arrows in F and I) with the GFP fusions on one side (green), the ST52-RFP on the other side (red) and a region of overlap between them (yellow).
  • Figure 8 shows a comparison of cytosolic tail and TMD length for plant N-glycosylation enzymes:
  • the position and the size of the transmembrane domain were estimated from the TmHMM_v2 software (http://www.cbs.dtu.dk/services/TMHMM/).
  • the probability to define the position of the TMD is below to 50% (//).
  • Boxes outlined in bold correspond to N-glycan processing enzymes whose intracellular localization has been studied to date by confocal and/or electronic microscopy.
  • GCSI glucosidase I, ⁇ 1,2 ManI: ⁇ 1,2-mannosidase I, ⁇ 1,2 GNTI: , ⁇ 1,2-N-acetylglucosaminyltransferase I, ⁇ 1,3 ManII: ⁇ 1,3-mannosidase II.
  • ⁇ 1,2 GNTII ⁇ 1,2-acetylglucosaminyltransferase II
  • ⁇ 1,2 XylosylT ⁇ 1,2-xylosyltransferase
  • ⁇ 1,3 FucT ⁇ 1,3-fucosyltransferase
  • ⁇ 1,4 FucT ⁇ 1,4-fucosyltransferase
  • ⁇ 2,6 sialylT ⁇ 2,6-sialyltransferase.
  • Figure 9 shows localization into the Golgi and the ER of a series of GFP fusions to truncated forms of Manl ( ⁇ 19Man, ⁇ 19Man49, figure 3 ) and to MAAAMan49, after stable expression (3-4 days) in tobacco BY-2 cells or transient expression (5 days) in tobacco leaf epidermal cells by leaf infiltration, using confocal laser scanning microscopy:
  • Figure 10 shows localization into the Golgi compartments of a series of GFP fusions to ManTMD23, Man99TMD23 or XylT35 ( figure 3 ) and the trans Golgi marker ST52-mRFP, after stable co-expression (3-4. days) in tobacco BY-2 cells of one or other of these GFP fusions and ST52-mRFP, using confocal laser scanning microscopy:
  • Figure 11 shows localization in the Golgi apparatus of GFP fusions to Man99 and Man99TMD23, using electron microscopy coupled to immunogold-labeling with polyclonal anti-GFP antibodies from suspension-cultured BY2 tobacco cells:
  • SV secretory vesicle.
  • Figure 12 shows localization into the Golgi and the ER of a series of GFP fusions to members of the N-glycan processing machinery (ManI, XylT), truncated forms thereof (Man99TMD23, Man99, , figure 4 ) after transient expression (5 days) in leaf epidermal cells by leaf infiltration, using confocal laser scanning microscopy:
  • Figure 13 shows effects of a 2h treatment with BFA on ER and/or Golgi proteins in BY-2 cells expressing a soluble ER marker (GFP-HDEL, A-B), a membrane ER marker (Glu90-GFP, C-D), an ER and early Golgi marker ( ⁇ 19Man49-GFP, E-F), a medial Golgi marker (XyIT35-GFP, G-H), or late Golgi markers (Man99TMD23-GFP, ST52-mRFP, I-L), using confocal laser scanning microscopy.
  • GFP-HDEL soluble ER marker
  • Glu90-GFP Glu90-GFP, C-D
  • an ER and early Golgi marker ⁇ 19Man49-GFP, E-F
  • a medial Golgi marker XyIT35-GFP, G-H
  • late Golgi markers Man99TMD23-GFP, ST52-mRFP, I-L
  • Figure 14 shows effects of a 2h treatment with BFA (50mg.mL -1 ) on the simultaneous redistribution of both early and late Golgi markers into the ER and into the Golgi clusters, using confocal laser scanning microscopy, from:
  • FIG. 15 is a schematic representation of the constructs we used in this study: GCSI: full length A. thaliana ⁇ -glucosidase I fused to GFP / GCS150: the first 150 amino acids of GCSI fused to GFP / GCS90: the first 90 amino acids of GCSI fused to GFP / ⁇ 13GCS150: the 13 first N-terminal amino acids (MTGASRRSARGRI-SEQ ID N°1) were deleted from GCS150 / ⁇ 13GCS90: the 13 first N-terminal amino acids were deleted from GCS90/ Hs10- ⁇ 13GCS90: the 10 first N-terminal amino acids of Homo sapiens GCSI (MARGERRRRA-SEQ ID N°2) were fused at the N-terminus of ⁇ 13GCS90 / XYLT35: the first 35 amino acids of A.
  • GCSI full length A. thaliana ⁇ -glucosidase I fused to GFP / GCS150: the first 150 amino acids of G
  • GCSI fused to GFP / GCS13-XYLT35 the 13 first N-terminal amino acids of GCSI were fused to XYLT35 /CNX11-XYLT35: the last 11 amino acids (NDRRPQRKRPA-SEQ ID N°3) of A.
  • thaliana calnexin were fused to the N-terminus of XYLT35 /ST52-GFP/mRFP: the first 52 amino acids of a rat ⁇ -2,6-sialyltransferase (ST) were fused to GFP or mRFP/ GFP/mRFP-HDEL: GFP or mRFP under the control of the sporamine signal peptide and the HDEL ER retention sequence
  • CT cytosolic tail
  • TMD transmembrane domain
  • CD catalytic domain.
  • Figure 16 shows that GCSI accumulates strictly in the ER: Transgenic BY-2 tobacco cell lines were observed 3-4 days after sub-culturing. Cortical (A,C) and medial optical sections (B, D) show GCSI is located in the ER (A,B) and the pattern staining is similar to that of GFP-HDEL (C, D).
  • Figure 18 shows that the N-terminal sequence of 13 amino acids contains ER localization information.
  • GCS90 accumulates in the ER in BY-2 cells (A, B)
  • ⁇ 13GCS90 is located in the Golgi (C, D).
  • XYLT35 is a Golgi protein (E, F)
  • GCS13-XYLT35 is found in the ER and in the Golgi (G, H).
  • Figure 19 shows that the Arg-rich ER targeting sequence is conserved between kingdoms Nicotiana tabacum leaf epidermal cells expressing ⁇ 13GCS90, XYLT35, GCS13-XYLT35, Hs10-GCS90 or CNX11-XYT35 alone (A, D, G, J, M), or with mRFP-HDEL (B, E, H, K, N), or with ST52-mRFP (C, F, I, L, O).
  • ⁇ 13GCS90 (A-C) is exclusively in the Golgi whereas GCS90 was in the ER and ⁇ 13GCS90 perfectly co-localises with ST-mRFP (C, F).
  • the Hs10-GCS90 chimeric protein is in the ER only (J) and co-localises with mRFP-HDEL (K) but not with ST52-mRFP (L). This suggests signals are conserved between kingdoms.
  • mRFP-HDEL mRFP-HDEL
  • the ER appears in yellow and the Golgi remains green (N) whereas with ST52-mRFP the Golgi is yellow and the ER is green (O) showing GCS13-XYLT35 also has a dual location in the ER and in the Golgi.
  • Figure 20 shows that the N-terminal arginine contains ER localization information Nicotiana tabacum leaf epidermal cells expressing GFP fusions alone (left panel), or with mRFP-HDEL (middle panel) or with ST52-mRFP (right panel).
  • GCS90 (A) perfectly co-localises with mRFP-HDEL (B, ER in yellow) but not with ST52-mRFP (C, ER in green, Golgi in red).
  • D-F Ala
  • G-I Leu
  • R/L6-7GCS90 J- L
  • R/L10-12GCS90 M-O
  • R/L6-L12 P-R
  • K, N, Q ER soluble protein mRFP-HDEL
  • Golgi stacks L, O, R
  • Figure 21 shows that R/L6-7 are closely associate to the Golgi.
  • R/L6-7GCS90 are co-expressed with mRFP-HDEL and ST52-mRFP, the fluorescent structures appear in yellow showing their very close association with the Golgi.
  • Figure 22 shows that Nicotiana tabacum leaf epidermal cells expressing GFP-fusions with Sar1p-mRFP (panels B, E and K) or Sar1p-GTP-mRFP (panels H and N).
  • Arg are mutated by pairs
  • R/L6-7GCS90 (A-C) or GCS90 (D-I) are co-expressed with mutated or not mutated Sar1p, no labelling modification was observed for GFP-fusions.
  • the co-expression of R/LGCS90 with Sar1p -GTP blocks the transport of R/LGCS90 in the ER while the co-expresion with Sar1p doesn't modify the pattern of R/LGCS90.
  • FIG. 23 shows that the N-terminal arginine motifs are not the key determinant for ER retention of At GCSI.
  • ER is visualized in transient GCS150-GFP (A), GCS90-GFP (B), and ⁇ 13GCS150-GFP (C) transformed Nicotiana tabacum leaf epidermal cells, cortical section.
  • ER localization of ⁇ 13GCS150-GFP is confirmed by co-expression with mRFP-HDEL (E) and ST52-mRFP (G), cortical sections.
  • E mRFP-HDEL
  • G ST52-mRFP
  • Figure 24 shows that the luminal targeting determinant is sufficient to retain XYLT35 in the ER:
  • Figure 25 shows localisation analysis of GFP fusion in BY-2 cells by confocal microscopy; Cortical (A) or transversal (B) View. Signals recently identified in our group have been fused to GFP and the localisation of the recombinant protein has been analyzed after stable expression in BY-2 tobacco cells. Fusion between GFP and signals 1, 2, 3 or 4 highlighted the ER like the GFP-HDEL fusion (panels A-F). Fusion between GFP and signals 5, 6, 7, 8 or 9 highlighted the ER but also aggregates assimilated to Golgi clusters, like the control ManI-GFP (panels G-L).
  • Figure 26 shows the localization of recombinant proteins harboring a di-arg motif.
  • Recombinant protein harboring one or two di-arg motifs colocalized with the mRFP-HDEL ER maker (A-C; G-R) while the mutations of arginine are responsible of the co-localisation of the recombinant protein with ST52 -mRFP Golgi marker ( D-F).
  • Figure 27 shows localization of targeting signals on the type I or type II membrane protein.
  • FIG 28 illustrates that an antibody or an antibody fragment, specific for a membrane protein and fused with one of the targeting sequence described here has the capacity to target the membrane protein in a different compartment of the secretory pathway.
  • a GFP localized in the Golgi is used for illustration.
  • Construction of plasmids and some examples of plasmids used for targeted expression of a scFv are presented panels A and B.
  • panel C a membrane protein localized in the Golgi (GFP-golgi) when expressed alone is reoriented to an ER/Golgi compartment when co-expressed in a same plant cell with a GFP-specific scFv fused with SEQ ID NO: 33.
  • Figure 29 illustrates that an heterologous enzyme, here human beta 1,4 galactosyltransferase, can be targeted in the different compartments of the plant secretory pathway after fusion with one of the targeting signals described herein.
  • panel B illustrates plasmids used for expression of fusions of human ⁇ 1,4 galactosyltransferase with SEQ ID NO: 8, 33, 36, or 38 in plant cells.
  • Figure 30 illustrates that targeted expression of human beta 1,4-galactosyltransferase after fusion of this glycosyltransferase catalytic domain with SEQ ID NO: 36 strongly improves the efficiency of this heterologous glycosyltransferase (panel B) when compared to glycosylation patterns obtained when the same glycosyltransferase is targeted in the plant secretory pathway by its own human targeting sequence (panel A).
  • Figure 31 presents one of the plasmids prepared to accumulate a glycan maturation enzyme in protein bodies generated by the ZERA® peptide.
  • the plasmid detailed here allows expression of mannosidase fused with Zera® in a plant cell.
  • EXAMPLE A ADRESSING PROTEINS TO THE ER AND/OR GA (ANNEXED FIGURE 3A)
  • ManI-GFP full-length GFP fusion
  • a linker containing an AatII restriction site was introduced between the ManI and the GFP coding regions. In combination with the native AatII site near the end of the predicted stem region this allowed for simple removal of the catalytic domain to yield Man99-GFP.
  • a longer TMD region was introduced in a two-step PCR mutagenesis of the modified Manl described above.
  • the AatII site following the TMD was replaced with a BspEl site.
  • a long PCR primer was used to duplicate the last seven aa of the predicted TMD to yield ManTMD23-GFP.
  • the catalytic domain of this construct was removed with AatII to give Man99TMD23-GFP.
  • ST-mRFP To obtain the plant binary vector encoding ST-mRFP, GFP is replaced with monomeric RFP (provided by Roger Tsien) in pVKH18En6 ST-GFP ( Saint-Jore et al., 2002 ( ref . 56). ST-mRFP expression is under control of 6x tandemly-repeated CaMV 35S promoters.
  • the GNTI-GFP, the GNT38-GFP were amplified by PCR using the N. tabacum cDNA encoding N-acetylglucosaminyltransferase as template (Strasser et al, 1999 ( ref . 60)).
  • GCSI-GFP the total cDNA was amplified by PCR using A.
  • the 90 aa deleted of the first 13 aa were amplified by PCR with forward (5'-CGGGGTACCCCATGAAATCATCATCATTATCTCCC-3' (SEQ ID N°49)) and the same reverse primers as above to give D13GCS90-GFP.
  • Fluorescence of a full-length ManI-GFP fusion construct was detected by confocal laser scanning microscopy in small bodies (annexed figures 5A and 5B ) that moved through the cytoplasm as it has been described previously for this construct in another independent cell line (Nebenpublished et al., 1999 ( ref . 43)).
  • the full length protein (Boisson et al., 2001 (ref.
  • the second candidate investigated was (GNTI) from Nicotiana tabacum (Strasser et al., 1999 (ref. 60)).
  • This glycosyltransferase adds the first N-acetylglucosamine residue on N-glycans soon after Manl has removed an a-1,2-mannose (annexed figure 2B ).
  • the full length protein was fused to GFP and GNTI-GFP was expressed in tobacco BY-2 suspension-cultured cells.
  • the steady state location of the fusion was the Golgi and the ER (annexed figure 5H ) in a pattern very similar to ManI-GFP (compare annexed figures 5H and 5B ).
  • the third candidate, b-1,2-xylosyltansferase (XylT) from Arabidopsis was located in the Golgi only (annexed figure 5 ), confirming the results from Pagny et al. (2003) ( ref. 50) who demonstrated that the N-terminal end of this enzyme targets GFP to a medial subset of cisternae of Golgi stacks.
  • N-glycosylation enzymes are targeted specifically to the ER (GCSI) or to the Golgi (XylT) exclusively, but some enzymes have a dual steady state location in both organelles as it is the case for the Manl and the GNTI and other membrane proteins such as prolyl 4-hydroxylase (Yuasa et al ., 2005 ( ref . 65) and ERD2 (Boevink et al., 1998 ( ref . 5); Saint-Jore et al., 2002 ( ref . 56)).
  • the first 99 aa (CT+TMD+S) or the first 49 aa (CT+TMD) of Manl were fused to GFP and the corresponding chimeric proteins were named Man99-GFP and Man49-GFP, respectively (annexed figure 4 ).
  • Man99-GFP and Man49-GFP were either stably expressed in BY-2 suspension cultured cells or transiently expressed in tobacco leaf epidermal cells by leaf infiltration.
  • ManI-GFP was co-expressed with the trans Golgi marker ST52-mRFP which is derived from ST52-GFP ( Saint-Jore et al., 2002 ( ref . 56); Runions et al., 2006 ( ref . 55) by replacing GFP with the monomeric red fluorescent protein (mRFP, Campbell et al., 2002 (ref. 11 ).
  • the first 77 N-terminal aa of the tobacco GNTI including the CT, the TMD and the stem, were previously described to contain the information required to maintain Golgi retention of this glycosyltransferase (Essl et al. 1999 (ref. 16)).
  • This polypeptide domain fused to GFP has been shown to be preferentially located in the Golgi but the chimeric protein was also detected in the ER as observed here for the full length construct (annexed figure 5H ).
  • the lumenal part (39 aa) was removed and the remaining first 38 N-terminal aa (CT+TMD) of this glycosyltransferase were fused to GFP (annexed figure 4 ).
  • the fusion protein was named GNT38-GFP and stably expressed in BY-2 suspension cultured cells or transiently in tobacco leaf epidermal cells.
  • GNT38-GFP was located in the Golgi and in the ER (annexed figure 6G and 6H ) as previously observed for the full length construct (GNTI-GFP, annexed figure 5H ).
  • GNT38-GFP was stably co-expressed with ST52-mRFP in BY-2 cells, as observed before with Man99-GFP and Man49-GFP, the two fluorescents spots overlapped, but some red fluorescence was distinguishable from the yellow suggesting GNT38-GFP is not in the trans-Golgi (annexed figures 7J-L ) as previously observed for ManI.
  • the N-terminal cytosolic region of many membrane bound proteins residing in the mammalian and yeast ER and/or in the Golgi apparatus contains signals which facilitate either their retrieval from the Golgi back to the ER (Teasdale and Jackson, 1996 ( ref . 62); Zerangue et al., 1999 ( ref . 66)) or their export from the ER to the Golgi (Giraudo and Maccioni, 2003 ( ref . 20).
  • the length of cytoplasmic tails can vary widely between the different glycosidases and glycosyltransferases (annexed figure 8 ). For instance, GCSI and Manl contain a long cytoplasmic tail (51/29 aa respectively). In comparison, the CTs of GNTI and XylT are only composed of 11 aa.
  • CT sequence was substituted by an artificial sequence MAAA, (MAAAMan49-GFP, annexed figure 4 ). This artificial sequence does not contain any known targeting sequences and does not affect the length of the hydrophobic transmembrane domain.
  • the three constructs, MAAAMan49-GFP, D19Man-GFP and D19Man49-GFP have been expressed in tobacco cells.
  • the two latter labelled the ER and the Golgi (annexed figures 9A-D ) just like the constructs they originate from (annexed figure 5A, 5B and 6D, 6F respectively).
  • the fusion protein containing an artificial MAAA CT was located in the same compartments (annexed figure 9E ) indicating that the N-terminal cytosolic region is not necessary for Manl targeting and consequently all information required for its steady state localization to both the ER and the Golgi apparatus is contained within the 20 aa of the MAAAMan49-GFP construct i.e., in the TMD and C-terminal flanking amino acids.
  • TDM Trans Membrane Domain
  • the "kin recognition" model (Nilsson et al., 1993b ( ref . 45)
  • aggregation of N-glycan maturation enzymes by homo/hetero-oligomerization would prevent the resulting large complexes from being delivered to secretory vesicles and ongoing forward transport downstream in the secretory pathway.
  • the "kin recognition" model would have to be modified to allow for the oligomeric complexes to be preferentially packaged into retrograde vesicles (Füllekrug and Nilsson, 1998 (ref. 18)).
  • a second model, the lipid bilayer model proposes that the fit between the length of TMD of glycan maturation enzymes and the thickness of the lipid bilayer of each organelle membrane determines the localization because each organelle has its specific membrane lipid composition and consequently its own thickness (Hartmann and Benveniste, 1987 ( ref . 29); Lynch, 1993 ( ref . 33); Moreau et al., 1998 ( ref . 36); Morré and Mollenhauer, 1974 ( ref . 37)).
  • the distribution of N-glycan maturation enzymes in the secretory pathway is based on the length of their TMDs (Bretscher and Munro, 1993 (ref. 10)).
  • the membranes of the secretory pathway organelles increase in thickness from the ER to the plasma membrane.
  • the ER and the cis-Golgi membranes are only 4-5 nm thick whereas the membranes of the trans -Golgi, the secretory vesicles and the plasma membrane are 8-9.5 nm thick (Grove et al., 1968 ( ref . 26); Morré and Mollenhauer, 1974 ( ref . 37)).
  • TMD length was previously illustrated by studying the location of reporter proteins after varying the length of their TMD, in animal systems (Munro 1991, 1995a,1995b ( ref . 38)) and, for type I proteins, also in plant cells (Brandizzi et al., 2002a ( ref . 9)). This implies, the membrane of a specific compartment can only accommodate hydrophobic TMDs of the matching length
  • the human lysosomal membrane protein LAMP1 containing a 23 aa TMD was fused to GFP and was expressed in tobacco leaves. The fusion was located in the plasma membrane.
  • the TMD was shortened to 20 and 17 aa
  • the GFP chimeras were localized to the Golgi and ER membranes, respectively.
  • the 19 aa long TMD of the vacuolar sorting receptor BP80 targeted GFP to the Golgi whereas a lengthened TMD of 22 aa targeted GFP to the plasma membrane.
  • the TMD length of the type II membrane protein Manl was investigated in order to know how it could affect its sub-compartmentation in the Golgi.
  • the TMD length was increased from 16 to 23 aa by duplicating the seven last aa of this domain.
  • chimeric proteins with a 23 aa TMD were localized exclusively to the Golgi and more precisely in the trans -half of the Golgi stacks
  • the information required for Manl targeting is contained within a 20 amino acid (aa) sequence including the 16 aa TMD.
  • aa 20 amino acid sequence
  • two fusion proteins, ManTMD23-GFP and Man99TMD23-GFP were designed, where the TMD of Manl was lengthened from 16 to 23 aa by duplication of its last seven aa (annexed figure 4 ).
  • ManTMD23-GFP and Man99TMD23-GFP were expressed in BY-2 suspension cultured cells and in tobacco leaf epidermal cells.
  • ManTMD23-GFP and Man99TMD23-GFP were exclusively located in bright spots (annexed figure 10A, 10B, 10D ), sensitive to the fungal toxin brefeldin A (50 ⁇ g.mL -1 , 2h, annexed figure 11 C) .
  • ManTMD23-GFP and Man99TMD23-GFP were similar to either the XylT-GFP fusion (annexed figure 10 ), or the ST52-mRFP fusion (annexed figure 10 ) both located exclusively in the Golgi in BY-2 suspension cultured cells and tobacco leaf epidermal cells as it has been confirmed previously by electron microscopy (Boevink et al ., 1998 ( ref . 7); Pagny et al., 2003 ( ref . 63)).
  • the spot patterns were similar in cortical images (annexed figure 8D-F ) compared to cross sections (annexed figures 10G-I ) reinforcing the assumption the Man-GFP fusions with a longer TMD and the trans-Golgi marker ST52-mRFP perfectly co-localize.
  • the medial Golgi marker (XylT35-GFP) and the trans Golgi marker (ST52-mRFP) resulted in fluorescent spots that did not overlap perfectly in the merged image (annexed figure 10J-L ).
  • Electron microscopy coupled to immunogold-labeling with polyclonal anti-GFP antibodies allowed us to to determine more precisely the intra-Golgi localization of these fusion proteins.
  • the Man99-GFP fusion accumulated mainly to the cis- side of the Golgi (annexed figure 11 B) whereas the Man99TMD23-GFP fusions are principally localized to the trans -side of the Golgi (annexed figure 11 C) .
  • ManI-GFP and ManTMD23-GFP Similar results were obtained with ManI-GFP and ManTMD23-GFP (data not shown). Control experiments using the pre-immune serum or wild-type tobacco BY-2 suspension- cultured cells showed no or very little non specific Golgi labeling (annexed figure 11A ).
  • TMD length requirements for a membrane protein to stay in a membrane with a given thickness might depend on the topology of the protein (type I or type II).
  • Man99-GFP was located in the Golgi and ER in soybean (annexed figure 12A ) or in tomato (annexed figure 12C ), whereas Man99TMD23-GFP was found almost exclusively in the Golgi in both expression systems (annexed figure 12B and 12D ).
  • results obtained with GCSI illustrates that specific localization of some membrane proteins in the ER or Golgi membranes could also depend on both protein-lipid (via the TMD) and protein-protein (via special sorting motifs) interactions.
  • the identification of cytosolic partners such as Golgi matrix proteins or cytoplasmic regulators permit to explain mechanisms involved in this second model for partitioning the N-glycan maturation enzymes along the plant secretory pathway.
  • ER soluble or membrane markers (GFP-HDEL or Glu90-GFP, annexed figures 13A-D ), the ER /early Golgi marker (D19Man49-GFP, annexed figure 13E-F ), the medial Golgi marker (XylT35-GFP, annexed figure 13G-H ) or the late Golgi marker (Man99TMD23-GFP or ST52-mRFP, annexed figure 13I-L ) were treated with BFA (50mg.mL -1 ) during 2h.
  • BFA 50mg.mL -1
  • BFA induces the redistribution of both markers into the ER and into Golgi aggregates (annexed figure 14 ), except for GFP-HDEL (annexed figures 14 A-F ) and Glu90-GFP (annexed figure 14 D) that were not found in the aggregates.
  • GFP-HDEL annexed figures 14 A-F
  • Glu90-GFP annexed figure 14 D
  • there are subtle differences in timing but these are not trivial to detect and also not informative with respect to intra-Golgi localization.
  • no significative difference was observed in the fluorescent patterns observed after BFA treatment of cell expressing either a soluble (GFP-HDEL) or a membrane protein (Glu90-GFP) marker (annexed figures 14A-D ).
  • the N-terminal sequences of characterized glycosylation enzymes were compared (annexed figure 14 ).
  • GluI was selected to check for general applicability of this model.
  • TMD of Glul was sufficient for its targeting and retention in the ER, most of the luminal part of this glycosidase was deleted (containing the catalytic domain) and fused its first N-terminal 90 aa (CT + TMD + S) to GFP to get the fusion protein Glu90-GFP (annexed figure 4 ).
  • the first N-terminal 13 aa from the Glu90-GFP construct have been deleted to obtain D13Glu90-GFP (annexed figure4 ).
  • the chimeric protein was located exclusively in Golgi-like spots (annexed figures 15D and 15E) as observed for XylT-GFP (annexed figures 6I and 9E ) and ST52-GFP (annexed figure 15F).
  • the 18 aa long TMD of Glul is not sufficient to target this glycosidase in the ER membrane and additional information contained in the first 13 aa of the CT is required for the normal localization of this glycosylation enzyme in the secretory system.
  • GCSI accumulates strictly in the tobacco endoplasmic reticulum
  • Arabidopsis thaliana GCSI is a type II membrane protein, consisting of a 51 amino acid cytosolic tail, an about 18 residues transmembrane domain and a large catalytic domain directed toward the lumen (Boisson et al., 2001 (ref. 6)).
  • a GFP fusion to a full length GCSI was studied after stable expression in tobacco BY-2 cells (annexed figures 16A and 16 B; annexed figures 17 A and 17 B ).
  • Fluorescence of a full-length GCSI-GFP fusion construct was detected by confocal laser scanning microscopy exclusively in a reticulate network throughout the cytoplasm that was indistinguishable from the ER network stained by a GFP-HDEL construct (annexed figures 16 C and 16 D ).
  • the role of the luminal domain in GCSI targeting was first investigated.
  • the first 150 amino acids (aa) (CT+TMD+stem81aa) or the first 90 aa (CT+TMD+stem21aa) of GCSI were fused to GFP and the corresponding chimeric proteins were named GCS150 and GCS90, respectively (annexed in figure 15 ).
  • GCS150 and GCS90 were either stably expressed in BY-2 suspension cultured cells or transiently expressed in tobacco leaf epidermal cells by Agro-infiltration and were both observed in the ER in both expression systems (annexed in figure 17 A,C,E and 17B,D,F respectively) exactly as previously observed for the full length construct (annexed in Figure 16 A,B ). It is important to note that when these truncated fusions were transiently expressed in tobacco leaf epidermal cells, the ER labeling was still observed five days after transformation when the overall expression level is already strongly declining and whereas Golgi fusions analyzed in the same condition are located exclusively in the Golgi (Saint-Jore-Dupas et al., 2006; data not shown). This data show the catalytic luminal domain of glucosidase I is not necessary for the ER location of the enzyme and the first 90 amino acids of GCSI are sufficient to retain GFP in the ER
  • the N-terminal cytosolic region of many membrane proteins residing in the mammalian and yeast ER contains signals which facilitate either the strict retention (Nilsson et al., 1994 ( ref . 46); Opat et al., 2000 ( ref . 48), Hardt et al., 2003 ( ref . 28); Ciczora et al., 2005( ref. 12)), or their retrieval from the Golgi back to the ER (Teasdale and Jackson, 1996 ( ref .62); Zerangue et al ., 1999 ( ref .66)) whereas some others promote the export from the ER to the Golgi (Giraudo and Maccioni, 2003 ( ref .20)).
  • the first 13 amino acids located at the N-terminal end of GCS90 was first deleted and the resulting chimeric protein was named D13GCS90 (annexed in figure 15 ).
  • This truncation removed two potential dibasic motifs RR or RXR that might function in ER retention although other potential ER-retention signals (RR or KXK) remained in the CT of this fusion protein.
  • the first 13 N-terminal amino acids peptides of GCSI were fused to a Golgi reporter protein (XYLT35, annexed Figure 15 ) previously located in the medial Golgi (Pagny et al., 2003( ref . 50)).
  • XYLT35 was confirmed to be exclusively accumulated in the Golgi apparatus of BY-2 tobacco cells as it is illustrated in Figure 18 G to H .
  • the two constructs were also stably expressed in BY-2 cells.
  • the deletion of the first 13 amino acid of the cytosolic tail relocated the GCS90 protein into bright spots ( Figure 18C-D , Figure 19A ) similar to the one observed with the Golgi marker XYLT35 ( Figure 18E-F ).
  • the D13GCS90 did not colocalize with the ER marker mRFP-HDEL ( Figure 19B ) but perfectly colocalized with the ST-mRFP Golgi marker ( Figure 19C ) after transient expression in tobacco leaf epidermal cell.
  • the first 13 amino acid of the GCSI are necessary to retain the GCS90 fusion protein in the ER and are sufficient to relocate a Golgi marker in the ER.
  • Hs10-GCS90 the first N-terminal 13 amino acids of GCS90 was substituted by the first N-terminal 10 amino acids of human GCSI (Hs10-GCS90, Figure 15 ).
  • Hs10-GCS90 When transiently expressed in tobacco leaf epidermal cells, Hs10-GCS90 was located in the ER indicating that the N-terminal cytosolic region is perfectly recognized by the plant secretory system ( Figure 17J to L).
  • the next step was to identify the structure labeled as fluorescent spots by the R/L 6-7 GCS90, R/L 10-12 GCS90 and R/L 6-12 GSC90 chimeric fusion protein.
  • R/L 6/7 GCS90 proteins GFP fluorescence accumulated in the ER and in fluorescent structures which are smaller that Golgi stacks ( Figure 20 J to L and Figure 21 A to B ).
  • the mRFP-HDEL ER marker, the ST52-mRFP Golgi marker and either R/L 6-7 GCS90, R/L 10-12 GCS90 or R/L 6-12 GSC90 fusion proteins were expressed simultaneously.
  • the R/L 6-7 GCS90, R/L 10-12 GCS90 and R/L 6-12 GSC90 fusion proteins were accumulated in the ER in small intermediate domains located between the ER and the Golgi, from which ER resident soluble proteins are excluded ( Figure 20K and 20N ). These domains are strongly associated with the Golgi and move with the Golgi stacks along the ER cortical network, so these domains could be ER-exit-sites (ERES) as described in Yang et al., 2005.
  • ERES ER-exit-sites
  • the transport of R/LGCS90 is regulated by the cytosolic protein Sar1p.
  • the small domains labeled with fusion proteins harboring one RR, RXR or RXXR motif are not associated with the Sar1p-mRFP although they are located between the ER and the Golgi compartments.
  • the N-terminal 13 aa were deleted from the full-length sequence of the GCSI to evaluate the significance of the motifs (RXR and RR) in ER targeting of wild type enzyme (D13GCSI, Figure 15 ).
  • the GCS150 truncated form was synthesized lacking the amino acid residues 1-13 at the N-terminal end, (D13GCS150, Figure 15 ). Fusion proteins were then expressed transiently in leaf epidermal cells.
  • both D13GCSI and D13GCS150 were exclusively located in the ER ( Figure 23D-E ).
  • mRFP-HDEL ER-marker
  • both D13GCSI and D13GCS150 were not detected in the Golgi and both fluorescence signals were observed in the ER and perfectly co-localized with mRFP-HDEL ( Figure 23G-L ).
  • the 81 amino acid residues 70-150 or the 61 aa 90-150 were fused to the luminal domain of the medial Golgi marker XYLT35 ( Figure 24A ) and transiently expressed these new proteins in tobacco leaf epidermal cells (XYLT35-GCS81 and XYLT35-GCS60, Figure 24B,C ). Most of the GFP labeling was found in the ER, the "remnant" being in the Golgi.
  • ER residency can be accomplished by direct retention involving association of protein subunits to give large oligomeric complexes via their transmembrane and/or luminal domains, as previously described in the kin-recognition model for Golgi-located membrane proteins. (Nilsson et al., 1994 (ref. 46); Opat et al., 2000 (ref. 48)). These large protein oligomers were assumed to escape packaging into transport vesicles, thus preventing their export from the organelle. This type of mechanism may be functional in the ER retention of subunit components of the hetero-oligomeric oligosaccharyltransferase complex but were not described in plants yet.
  • both the cytosolic tail and the luminal domain of AtGCSI contain ER targeting determinants Consistent with its specificity and in complete agreement with observations made in other eukaryotic systems, the results demonstrante that AtGCSI is localized in the plant ER and exclusively in this compartment.
  • Other glycosylation enzymes acting early in the plant N-glycan maturation such as N-acetylglucosaminyltransferasel (GNT1) and a1,2 mannosidase (ManI) have been shown to be located both in the ER and in the cis -Golgi .
  • the GNTI and ManI, ER and cis -Golgi have been shown to contain targeting information resides in the cytosolic tail whereas the first 13 N-terminal amino acids of GCSI cytosolic tail contain ER targeting information.
  • the present example shows which were the key amino-acids in the cytosolic tail involved in ER targeting but also demonstrates that an arginine-rich cytosolic tail is not the only ER targeting determinant in the whole protein sequence.
  • the deletion of the arginine-rich sequence from GCS150 does not permit the exit of the ER to a later compartment such as the Golgi and D13GCS90 is still exclusively located in the ER.
  • the cytosolic tail of GCSI contains three di-arginine signals independently sufficient for ER retention
  • MTAGASRRSARGRI SEQ ID N°1
  • Di-arginine motifs have been extensively studied in mammalian membrane proteins but they were never characterized before the present study in their plant homologues. Interestingly a di-arginine motif previously identified in human GCSI has been shown to mediate ER retention in plant cell (Hardt et al., 2003 (ref. 28)).
  • di-arginine motifs identified in AtGCSI look more flexible than their human homologue. Indeed the di-arginine motif of human a glucosidase I is made of two arginine residues in position +7 and +8 and of a basic amino acid in position +9. In AtGCSI the distance between two arginine residues looks more flexible but cannot exceed two amino acids for a good efficiency. Furthermore this motif should be in a close proximity of the N-terminal end of the protein. Indeed, in GCSI a di-arginine motif RR in position +23 and +24 is still present in the fusion protein D13GCSS90-GFP but is not sufficient to confer ER retention.
  • each signal listed in Table 3 is sufficient to target a reporter protein such as the green fluorescent protein to the ER and/or the GA (see annexed figures 25A and 25B ).
  • Table 3: LOC Localisation of reporter protein when fused to the signal (cf annexed figure 1) Signal Sequence LOC.
  • SEQ ID N°1 to 3 represents a set of anchoring sequences for membrane protein targeting to the ER. These sequences are located in the cytosolic tail of the membrane protein located at the C- or N-terminal end.
  • these di-Arg motif can be located at the N- or C-terminal part of respectively, the type II or type I membrane protein
  • SED ID N° 4 to 7 are responsible of a strict retention of the recombinant membrane polypeptide in the ER. These sequences are located in the ER lumen.
  • SEQ IDn° 4 to 7 located in the stem (S3) is responsible for a strict retention of membrane proteins in the ER.
  • S3 is a sequence located near the transmembrane domain.
  • a strict retention of the recombinant polypeptide in the ER is done by the conjunction of one of the SEQ ID N° 1 to 3, containing the Di-Arg motif, and one of the SEQ ID N°4 to 7, as illustrated in the SEQ ID N°8, and is responsible of membrane protein strict retention in the ER and stabilization of recombinant proteins.
  • a transmembrane domain (GS2) of from 16 to 23 amino acids has been shown to be sufficient to address a protein to the Golgi. The use of this domain is sufficient to anchor a recombinant protein or an enzyme in the Golgi membranes.
  • Examples of tested transmembrane domains (GS2) that are included in the peptidic signal are as following: signal SEQUENCE SEQ IDn° 16 XXXLALLFIVFVCVSFVFWDR cis 9 20 XXRYLLILAAVAFIYIQMRLFATQS cis 10 18 XXXLGILFAVTLSIVLMLVSVXXX 11 19 XXKIFLYMLLLNSLFLIIYFVFH median 12 21 XXXRKLSNLLPLCVALVVAEIGFLG cis 13 21 XXXRKVSTFLPICVALVVIIEIGFLC median 14 23 XXFNTITITIMIAFTFFLLFLTGFLQFXX trans 15 23 XXKRLALLFIVFVCVSFVFWCVSFVFWDR trans 16
  • peptide signal issued from the GS3 domain are the followings: SEQ ID n° GS3 24 25 26 27 28 29 30 31
  • Example 3 Prevention of the addition of immunogenic residue on N-glycans by storage of recombinant protein within the early secretory pathway compartment by using targeting sequence.
  • This strategy consists in the storage of recombinant proteins within the ER, i.e., upstream of Golgi cisternae, where immunogenic glyco-epitopes are added to maturing plant N-glycans. It was first shown that the addition of H/KDEL amino acid sequences at the C-terminal end of a recombinant soluble protein is sufficient for its retention in the plant ER (Gomord et al., 1997 ( ref . 24), 1999 ( ref . 22)).
  • KDEL-ER signal sequence was fused to both heavy and light chains of the antibody of two different antibodies.
  • sequence SEQ ID N° 1 to 8 have been used to target heavy and light chain of the antibodies to the ER.
  • sequence SEQ ID N° 32 to 36 have been used to target heavy and light chain of antibodies to the ER and GA.
  • EXAMPLE B EXPRESSION OF AN ANTIBODY OR AN ANTIBODY FRAGMENT IN THE ER AND/OR THE GA (ANNEXED FIGURE 15, LANE B)
  • an antibody or an antibody fragment in the ER and/or in the GA offer different strategies to improve recombinant protein production. For example it can lead to target non modified (mutated) therapeutical proteins to the ER and/or to the GA.
  • EXAMPLE C EXPRESSION OF A HOMOLOGOUS OR HETEROLOGOUS ENZYME IN THE ER AND/OR GA OF PLANT CELLS AND EXPRESSION OF RECOMBINANT PROTEIN IN SAID CELLS:
  • Example 1 Humanization of N-glycosylation in plant cells (annexed figure 15, lane C) by addressing mammalian glycosyltransferases in the ER/GA of plant cells.
  • N-glycosylation starts in the ER, with a cotranslational addition of an oligosaccharide precursor (Glc3Man9GlcNAc2) to specific asparagine residue on the nascent polypeptide.
  • Glc3Man9GlcNAc2 an oligosaccharide precursor
  • the oligosaccharide undergoes several maturations resulting in complex N-glycan.
  • Many pharmaceuticals including antibodies used for their effector functions, such as the triggering of the immune response (Wright and Morrison, 1994 ( ref . 64)), require glycosylation for their in vivo activity and stability. This is why use the potential plants can offer for the production of recombinant antibodies, it becomes necessary to inhibit these plant-specific maturations in order to obtain 'humanized' non-immunogenic N-glycans.
  • the human beta(1,4)-galactosyltransferase was fused with the golgi targeting signals.
  • Plasmids for hGalT and GNTIhGalT expression were assembled from pBLTl121 (Pagny et al., 2003 ref 50).
  • the CaMV 35S promoter was replaced- by the alfalfa plastocyanin promoter at Hindlll-Xbal sites.
  • the human ⁇ (1,4)-galactosyltransferase (hGalT) gene (UDP galactose: ⁇ -N-acetylglucosaminide: ⁇ (1,4)-galactosyltransferase; EC 2.4.1.22) was isolated from pUC19-hGalT with EcoRl digestion.
  • the 1.2 kb hGalT fragment was cloned into pBLTl221 at Smal sites.
  • a flag tag was then fused to the C-terminal end of the coding region by PCR using the FGalT forward (5'-GACTCTAGAGCGGGAAGATGAGGCTTCGGGAGCCGCTC-3' SEQ ID N° 92) and the reverse RGalTFlagStu (5'-AAGGCCTACGCTACTTGTCATCGTCATCTTTGTAGTCGCACGGTGTCC CGAAGTCCAC-3' SEQ ID N° 93) primers.
  • R622 was then produced by cloning this Xbal-Stul fragment into the binary vector pBLTl121 under control of Plasto promotor and Nos terminator.
  • the first N-terminal 38 a.a. from N. tabacum N- acetylglucosaminyltransferase I (GNTI) corresponding to the transmembrane domain were amplified by PCR using the forward FGNT (5'-ATCGAAATCGCACGATGAGAGGGTACAAGTTTTGC-3' SEQ ID N° 94) and reverse RGNTspe (5'-CCCATGATGCGATCTGCATATTCTGACTGTGTCGC-3' SEQ ID N°95) primers and successively cloned into pGEM-T vector, and into pBLTl221 by Apal/BamHl.
  • forward FGNT 5'-ATCGAAATCGCACGATGAGAGGGTACAAGTTTTGC-3' SEQ ID N° 94
  • reverse RGNTspe 5'-CCCATGATGCGATCTGCATATTCTGACTGTGTCGC-3' SEQ ID N°95 primers and successively cloned into pGEM
  • PCR amplification was done from pUC19-hGalT to eliminate its own TMD and create Spel and Stul sites.
  • the forward FGalTspe (5'-GGACTAGTGCACTGTCGCTGCCCGCCTGC-3' SEQ ID N°96) and reverse RgalTFlagStu (5'-AAGGCCTACGCTACTTGTCATCGTCATCTTTGTAGTCGCACGGTGTCC CGAAGTCCAC-3' SEQ ID N°97) were used to amplify the Spel/Stul hGalT fragment.
  • Well-established plants resulting of embryo development of responsive explants were transferred into soil in the greenhouse and leaves were analyzed.
  • N-linked glycans isolated from wild-type and GAlT or GNTI/GalT-transformed alfalfa plants was done as follow.
  • Proteins were extracted for 30 minutes at 4°C from 500 mg of fresh alfalfa leaves in 5 mL of extraction buffer (0.7 M Saccharose, 0.5 M Tris, 30 mM HCl, 0.1 M KCI, 2% beta-mercaptoethanol). Insoluble materiel was eliminated by centrifugation during 10 minutes, 5,000 g, at 4°C. The resulting supernatant is treated by adding 1 volume of water saturated phenol, during 30 minutes at 4°C. Then, proteins and glycoproteins contained in the phenolic fraction were precipitated, overnight, at -20°C, by 5 volumes of PB (0.1 M Ammonium acetate dissolved in Methanol).
  • extraction buffer 0.7 M Saccharose, 0.5 M Tris, 30 mM HCl, 0.1 M KCI, 2% beta-mercaptoethanol.
  • the proteins and glycoproteins were digested by successive treatments with pepsin and PNGase A as previously described in Bakker et al ., 2001. Then, the N-glycans were fluorescent labelled by 2-Amino Benzamide (2-AB).
  • MALDI-TOF mass spectra of these derivatized N-glycans were acquired on a Voyager DE-Pro MALDI-TOF instrument (Applied Biosystems, USA) equipped with a 337-nm nitrogen laser. Mass spectra were performed in the reflector, delayed extraction mode using 2, 5-dihydroxybenzoic acid (Sigma-Aldrich) as matrix.
  • the matrix freshly dissolved at 5 mg.mL -1 in a 70:30 % acetonitrile / 0.1% TFA, was mixed with the solubilized oligosaccharides in a ratio 1:1 (V/V). These spectra were recorded in a positive mode, using an acceleration voltage of 20,000 V with a delay time of 100 ns. They were smoothed once and externally calibrated using commercially available mixtures of peptides and proteins (Applied Biosystems).
  • N-glycosylation Human Beta (1,4) galactosylation Yeast Mannosyltransferase OCH1p Human GNT III O-glycosylation N-acetylglucosaminyl transferase Galactosyltransferase Proteolytic cleavage Serine proteases Cyteine proteases Amidation Oxygenese Lyase Phosphorylation Phosphorylase Gamma-carboxylation Gamma carboxylase Proteoglycan modif Glycotransferase Glycosidase Sulfation Sulfatase Hydroxylation Hydroxylase Acetylation Acetylase Cell wall polysaccharides modif. Glycotransferase Glycosidase Glycosidase
  • the present invention provide a method for producing protein, for modifying expressing protein in subcompartment of plant cells, for expressing heterologous proteins in the RE and/or GA of plant cells.
  • the invention provides also post transcriptional modified proteins.
  • EXAMPLE D EXPRESSION OF ZERA® PROTEIN FUSED WITH SIGNAL SEQUENCE.
  • a fused protein comprising ZERA® and sequence signal (SEQ ID N° 8) and mannosidase I was made in order to accumulate the glycosidase as a membrane protein in the protein bodies.
  • the targeting signal (SEQ IDn°8) was used to accumulate the enzyme in the membrane ER and allow the production of protein bodies by forming aggregates via the ZERA peptide.
  • Plasmide Construct The ADNc encoding the ZERA fused to the human mannosidase I (Acess N°Q9UKM7) was amplified by PCR and sub-cloned in the pBLTl121 containing the targeting signal (SED IDn°8) at the SPel and SacI endonucleases sites.
  • pVKH18En6-mRFP, PBLTI121-GFP and pBIN20-GFP-fusions were transferred into Agrobacterium tumefaciens (strain GV3101 pMP90, Koncz and Schell, 1986) by heat shock.
  • Transgenic Agrobacterium were selected onto YEB medium (per liter, beef extract 5 g, yeast extract 1 g, sucrose 5 g, MgSO 4 -7H 2 O 0.5 g) containing kanamycin (100 mg.mL -1 ) and gentamycin (10 mg.mL -1 ) and were used to transform Nicotiana tabacum (c.v. Bright Yellow-2) BY-2 cells, as described in Gomord et al., 1998.
  • Transformed tobacco cells were selected in the presence of kanamycin (100 mg.mL -1 ) for PBLTI121-GFP and pBlN20-GFP-fusions or hygromycin (40 mg.mL -1 ) for pVKH18En6-mRFP and cefotaxime (250 mg.mL -1 ).
  • kanamycin 100 mg.mL -1
  • hygromycin 40 mg.mL -1
  • pVKH18En6-mRFP and cefotaxime 250 mg.mL -1
  • microcalli were first selected onto kanamycin plates, and were then transferred onto hygromycin plates. After screening, calli expressing the GFP and or mRFP-fusions were used to initiate suspension cultures of transgenic cells. 3-4 days old BY-2 suspension-cultured cells were used for experiments.
  • the fused proteins made in the present examples was express in the transformed cells and accumulate the enzyme in the membrane ER and allow the production of protein bodies by forming aggregates via the ZERA peptide.
  • the present inventio permit to target proteins to specific domain of cells, to increase the yield of production of recombinant polypeptides, to prevent immunogenicity of recombinant polypeptides and to obtain therapeutically active recombinant polypeptides that are the exact copy of their natural counterpart. It also permits to produce post transcriptional modified proteins

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Claims (22)

  1. Ein Aminosäuresequenz aus einer Gruppe bestehend aus SEQ ID NO 1, SEQ ID NO 4 und SEQ ID NO 8.
  2. Ein rekombinantes Protein, das eine Aminosäuesequenz nach Anspruch 1 und ein Protein umfasst, wobei die Aminosäuresequenz mit dem C-Terminus oder dem N-Terminus des Proteins verschmolzen ist.
  3. Rekombinantes Protein nach Anspruch 2, wobei das Protein aus einer Gruppe ausgewählt wird, die ein Enzym, einen Antikörper oder einen Teil hiervon, ein Reporter-Protein, ein rekombinantes Protein, ein therapeutisch aktives Protein umfasst.
  4. Rekombinantes Protein nach Anspruch 2, wobei das Protein ein Enzym ist, das aus der Gruppe ausgewählt wird, die Glycosidase, Glycosyltransferasen, Protease, Kinase, Decarboxylase, Epimerase, Nukleotid-Zucker-Transporter umfasst.
  5. Eine Nukleinsäuresequenz, die eine Aminosäuresequenz nach Anspruch 1 oder ein rekombinantes Protein nach Anspruch 3 oder 4 codiert.
  6. Ein Nukleinsäurevektor, der die Nukleinsäuresequenz nach Anspruch 5 umfasst.
  7. Eine Pflanzenzelle, die mindestens eine Aminosäuresequenz nach Anspruch 1 und/oder ein rekombinantes Protein nach Anspruch 3 oder 4 und/oder einen Nukleinsäurevektor nach Anspruch 6 umfasst.
  8. Eine Pflanzenzelle nach Anspruch 7, wobei die Pflanzenzelle mindestens ein Protein nach Anspruch 3 oder 4 umfasst, wobei das Protein ein heterologes Protein ist.
  9. Eine Pflanze, die mindestens Folgendes umfasst: eine Aminosäuresequenz nach Anspruch 1 und/oder ein rekombinantes Protein nach Anspruch 3 oder 4 und/oder einen Nukleinsäurevektor nach Anspruch 6.
  10. Eine Pflanze nach Anspruch 9, wobei die Pflanze mindestens ein Protein nach Anspruch 3 oder 4 umfasst, wobei das Protein ein heterologes Protein ist.
  11. Eine Pflanze nach Anspruch 9 oder 10, wobei die Pflanze aus der Gruppe ausgewählt wird, die Alfalfa, Arabidospsis thaliana, Nicotiana tabacum, Glycine max, Lycopersicon esculentum, Solanum lycopersicum umfasst.
  12. Ein Verfahren zur Herstellung eines posttranslational modifizierten Polypetptids mit folgenden Schritten:
    - Transfektion oder Transformation einer Pflanzenzelle mit mindestens einem Nukleinsäurevektor nach Anspruch 6, wobei der Vektor ein rekombinantes Protein codiert, das ein Polypeptid vor der posttranslationalen Modifizierung oder ein im Bezug auf das Polypeptid anderes rekombinantes Protein ist;
    - Züchten der transfektierten Pflanzenzelle; und
    - Ernten des posttranslational modifizierten Polypeptids;
    wobei, wenn das rekombinante Protein sich von dem Polypeptid unterscheidet, das Verfahren ferner einen Schritt der Transkfektion der Pflanzenzelle mit mindestens einem das Polypeptid codierenden Nukleinsäurevektor umfasst.
  13. Verfahren nach Anspruch 12, In welchem das rekombinante Protein ein Polypeptid vor der posttranslationalen Modifikation ist.
  14. Verfahren nach Anspruch 12, in welchem das rekombinante Protein sich von dem Polypeptid unterscheidet und in welchem das rekombinante Polypeptid ein Antikörper oder ein Teil hiervon ist, der das Polypeptid spezifisch bindet oder erkennt.
  15. Verfahren nach Anspruch 12, in welchem das rekombinante Protein sich von dem Polypeptid unterscheidet und in welchem das rekombinante Protein ein endogenes oder heterologes Enzym ist, das an der posttranslationalen Modifizierung des Polypeptids beteiligt ist.
  16. Verfahren nach Anspruch 12, in welchem das rekombinante Protein sich von dem Polypeptid unterscheidet und in welchem das rekombinante Protein ein Antikörper oder ein Teil hiervon ist, das ein an der posttranslationalen Modifizierung des Polypeptids beteiligtes Enzym moduliert.
  17. Verfahren nach einem beliebigen der Ansprüche 12 bis 16, in welchem das Polypeptid mit einem Speicherprotein co-exprimiert wird.
  18. Verfahren nach einem beliebigen der Ansprüche 12 bis 17, in welchem die posttranslationale Modifizierung des Peptids in dem Membrankompartiment des endoplasmatischen Retikulums (ER) oder des Golgi-Apparats (GA) stattfindet.
  19. Verfahren nach einem beliebigen der Ansprüche 12 bis 18, in welchem das posttranslational modifizierte Polypeptid ein therapeutisch aktives Protein ist.
  20. Verfahren nach einem beliebigen der Ansprüche 12 bis 19, in welchem die Pflanzenzellen Zellen von Pflanzen sind, die aus der Gruppe gewählt werden, die Medicago sativa, Arabidospsis thaliana, Nicotiana tabacum, Glycine max, Lycopersicon esculentum, Solanum lycopersicum umfasst.
  21. Verwendung einer Aminosäuresequenz nach Anspruch 1, um die Retention eines mit der Aminosäuresequenz verschmolzenen rekombinanten Proteins in einer im endoplasmatischen Retikulum membrangebundenen Form zu ermöglichen.
  22. Verwendung nach Anspruch 21, wobei das rekombinante Polypeptid ein Membranprotein ist und die Aminosäuresequenz die Verankerung des Membranproteins in dem endoplasmatischen Retikulum ermöglicht.
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WO2008056265A2 (en) 2008-05-15
US20110195452A1 (en) 2011-08-11
AU2007318926A1 (en) 2008-05-15
AU2007318926B2 (en) 2013-05-02
KR20090079258A (ko) 2009-07-21
EP2089526A2 (de) 2009-08-19
MX2009004985A (es) 2009-10-08
RU2481399C2 (ru) 2013-05-10
WO2008056265A3 (en) 2008-08-28
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CN101622354A (zh) 2010-01-06
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